The major emphasis of the proposed continuation of the research project concerns the purification and the biochemical and immunologic characterization of neutral protease components of secretory granules from mast cells. Mast cells obtained from rat serosal surfaces and those derived from mouse hematopoietic tissue and maintained in culture, both of which can be obtained regularly in greatest abundance and purity, and from human pulmonary tissue, which are of greatest relevance to human biology, will be utilized. Specific aims with rodent mast cells include determination of the mechanisms by which exogenous chymase activates rat serosal mast cells and its relation to immunologic activation; the role of released chymase in the mast cell-mediated inflammatory response; purification and characterization of the chymotryptic and tryptic murine mast cell proteases; evaluation of the epitope in murine mast cells that is shared with human tryptase; and studies of the synthesis of neutral protease, histamine, and proteoglycan and their incorporation into secretory granules. Projects with human mast cells include development of conditions to improve the yield and purity of lung mast cells; production of monoclonal and monospecific antibodies against human mast cell tryptase to be used for immunoassays, tryptase subunit characterization, localization of tryptase in tissues and cells, and immunoaffinity chromatography of tryptase; and determination of the enzymatic properties of tryptase on synthetic and biologic substrates. In particular, biologic activities of tryptase on the coagulation, kinin, and complement pathways will be examined. Tryptase mays be a specific marker for mast cells in which case a specific and sensitive immunoassay for the enzyme could detect it in biologic tissues and fluids and thereby indicate mast cell involvement. An ability to determine mast cell involvement in human disease will assist with the diagnosis and eventual treatment of allergic diseases and in a better understanding of the biology of mast cells.